scholarly journals Characterization of a Novel Fructosyltransferase from Lactobacillus reuteri That Synthesizes High-Molecular-Weight Inulin and Inulin Oligosaccharides

2002 ◽  
Vol 68 (9) ◽  
pp. 4390-4398 ◽  
Author(s):  
S. A. F. T. van Hijum ◽  
G. H. van Geel-Schutten ◽  
H. Rahaoui ◽  
M. J. E. C. van der Maarel ◽  
L. Dijkhuizen
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Lactobacillus Reuteri ◽  
Bacterial Strains ◽  
Potential Health ◽  
Isolation And Characterization ◽  
A Cell ◽  
Encoding Gene ◽  
First Time

ABSTRACT Fructosyltransferase (FTF) enzymes produce fructose polymers (fructans) from sucrose. Here, we report the isolation and characterization of an FTF-encoding gene from Lactobacillus reuteri strain 121. A C-terminally truncated version of the ftf gene was successfully expressed in Escherichia coli. When incubated with sucrose, the purified recombinant FTF enzyme produced large amounts of fructo-oligosaccharides (FOS) with β-(2→1)-linked fructosyl units, plus a high-molecular-weight fructan polymer (>107) with β-(2→1) linkages (an inulin). FOS, but not inulin, was found in supernatants of L. reuteri strain 121 cultures grown on medium containing sucrose. Bacterial inulin production has been reported for only Streptococcus mutans strains. FOS production has been reported for a few bacterial strains. This paper reports the first-time isolation and molecular characterization of (i) a Lactobacillus ftf gene, (ii) an inulosucrase associated with a generally regarded as safe bacterium, (iii) an FTF enzyme synthesizing both a high molecular weight inulin and FOS, and (iv) an FTF protein containing a cell wall-anchoring LPXTG motif. The biological relevance and potential health benefits of an inulosucrase associated with an L. reuteri strain remain to be established.

Preparation and Characterization of the High Molecular Weight [3H]Hyaluronic Acid

1992 ◽  
Vol 57 (10) ◽  
pp. 2151-2156 ◽  
Author(s):  
Peter Chabreček ◽  
Ladislav Šoltés ◽  
Hynek Hradec ◽  
Jiří Filip ◽  
Eduard Orviský
Keyword(s):  
Molecular Weight ◽  
Hyaluronic Acid ◽  
High Molecular Weight ◽  
Methyl Bromide ◽  
High Performance ◽  
Hydrogen Atoms ◽  
Isolation And Characterization ◽  
Labelling Procedure ◽  
Enzymatic Depolymerization

Two methods for the preparation of high molecular weight [3H]hyaluronic acid were investigated. In the first one, hydrogen atoms in the molecule were replaced by tritium. This isotopic substitution was performed in aqueous solution using Pd/CaCO3 as the catalyst. In the second method, the high molecular weight hyaluronic acid was alkylated with [3H]methyl bromide in liquid ammonia at a temperature of -33.5 °C. High-performance gel permeation chromatographic separation method was used for the isolation and characterization of the high molecular weight [3H]hyaluronic acid. Molecular weight parameters for the labelled biopolymers were Mw = 128 kDa, Mw/Mn = 1.88 (first method) and Mw = 268 kDa, Mw/Mn = 1.55 (second method). The high molecular weight [3H]hyaluronic acid having Mw = 268 kDa was degraded further by specific hyaluronidase. Products of the enzymatic depolymerization were observed to be identical for both, labelled and cold biopolymer. This finding indicates that the described labelling procedure using [3H]methyl bromide does not induce any major structural rearrangements in the molecule.

scholarly journals Construction of a Novel Chimeric Dextransucrase Fused to the Carbohydrate-Binding Module CBM2a

2021 ◽  
Author(s):  
Reinaldo Fraga Vidal ◽  
Roberto Carlos Aristicas Ribalta ◽  
Lisandra Teresa Martínez Valdés ◽  
Meinardo Lafargue Gámez ◽  
Amanda Montes Alvarez ◽  
...  
Keyword(s):  
Binding Capacity ◽  
Structural Characteristics ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Molecular Weights ◽  
Isolation And Characterization ◽  
The Subject ◽  
Encoding Gene ◽  
First Time

The lactic acid bacteria (LAB) have great potential to produce homoexopolysaccharides (HoPS), have been the subject of extensive research efforts, given their health benefits and physicochemical properties. The HoPS functional properties are determined by structural characteristics of varied molecular weights, types of glycosidic linkages, degrees of branching and chemical composition. The dextransucrases (DSases) are responsible of the synthesis of a kind of HoPS (dextran polymers), which are among the first biopolymers produced at industrial scale with applications in medicine and biotechnology. The concept of glycodiversification opens additional applications for DSases. In that sense the design and characterization of new DSases is of prime importance. Previously, we described the isolation and characterization of a novel extracellular dextransucrase (DSR-F) encoding gene. In this study, from DSR-F, we design a novel chimeric dextransucrase DSR-F-∆SP-∆GBD-CBM2a, where DSR-F-∆SP-∆GBD is fused to the carbohydrate-binding module (CBM2a) of the β-1-4 exoglucanase/xylanase Cex (Xyn10A) of Cellulomonas fimi ATCC 484. This dextransucrase variant is active and without alteration in its specificity. The DSR-F-∆SP-∆GBD-CBM2a is purified by cellulose affinity chromatography for the very first time. Our results indicate that new hybrids and chimeric DSases with novel binding capacity to cellulose can be designed to obtain glyco-biocatalysts from renewable lignocellulosic materials.

scholarly journals The isolation and characterization of the high-molecular-weight glycoprotein from pig colonic mucus

1978 ◽  
Vol 173 (2) ◽  
pp. 569-578 ◽  
Author(s):  
T Marshall ◽  
A Allen
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Quaternary Structure ◽  
Water Soluble ◽  
Isolation And Characterization ◽  
Nuclease Digestion ◽  
Non Covalent Interactions ◽  
Covalent Interactions ◽  
Equilibrium Centrifugation

1. A high-molecular-weight glycoprotein constitutes over 80% by weight of the total glycoprotein from water-soluble pig colonic mucus. 2. It was isolated from from nucleic acid and non-covalently bound protein by nuclease digestion followed by equilibrium centrifugation in a CsCl gradient. 3. The glycoprotein has the following composition by weight: fucose 10.4%; glucosamine 23.9%; galactosamine 8.3%; sialic acid 9.9%; galactose 20.8%; sulphate 3.0%; protein 13.3%; moisture about 10%. 4. The native glycoprotein has the high mol.wt. of 15×10(6). 5. Reduction of the native glycoprotein with 2-mercaptoethanol results in a glycoprotein of mol.wt. 6×10(6). 6. Pronase digestion removes 29% of the protein (3% of the glycoprotein) but none of the carbohydrate. 7. The molecular weight of the Pronase-digested glycoprotein is 1.5×10(6), which is halved to 0.76×10(6) on reduction with 2-mercaptoethanol. 8. The contribution of non-covalent interactions, disulphide bridges and the non-glycosylated peptide core to the quaternary structure of the glycoprotein are discussed and compared with the known structure of pig gastric glycoportein.

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